Alternative Genetic Selection in Bacillus anthracis and Francisella tularensis

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DESCRIPTION (provided by applicant): Many of the category A select agent bacteria are naturally sensitive to antibiotics and thus introduction of antibiotic resistance selective markers for genetic studies should be avoided. Non-antibiotic selective markers are limited in their availability and have not been evaluated for use in these bacteria. Markers to be developed should have the characteristics of having the selective phenotype expressed when the gene is present in single copy in the organism, being stable when expressed on multi-copy plasmids, and not exerting polar effects on downstream genes when inserted into a multi-gene operon. Desirable properties also include that the selective agent employed should give a low background when sensitive organisms are plated under selective conditions, and the difference between the MIC concentration of sensitive versus resistant organisms should be large. Furthermore, it would be advantageous to be able to easily and precisely excise the selective marker to create a mutant strain lacking the marker cassette so that the antimicrobial markers can be used iteratively to create multiply mutated strains. In this project, we will evaluate heavy metal resistance markers, including resistance to arsenite, cadmium, chromate, and mercury as well as resistance to the herbicide bialaphos as potential selective markers for genetic analysis of Bacillus anthracis and Francisella tularensis. The minimal sequence required for selection in the two hosts will be determined and mutational studies will be carried out to optimize expression. The use of these markers on multicopy plasmids and single copy insertions will be validated in the two hosts. Construction of a transposon bearing these selective agents will be created and evaluated. Engineering of the selective marker cassettes flanked by loxP sites will be accomplished and use of a Cre recombinase-based system to excise the selection cassette from plasmid or chromosomal targets in these select agent bacteria will be evaluated.